Immunogenic compounds comprising peptides of IL1β

ABSTRACT

The present invention relates to peptides derived from the proinflammatory cytokines, interleukin-1β (IL1β) and tumor necrosis factor α (TNFα), and their use in human or veterinary therapy, such as to generally treat diseases linked to the overproduction of IL1β or TNFα as well as acute or chronic inflammatory diseases, rheumatoid arthritis, septic shock, autoimmune diabetes, graft rejection in the host, etc.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to new peptides derived from theproinflammatory cytokines Interleukin-1 beta (IL1β) and Tumor NecrosisFactor alpha (TNFα) and their use in human or veterinary therapy. Thediseases targeted by these therapeutic uses can be in particularrheumatoid polyarthritis, septic shock, auto-immune diabetes, graftrejection in the host, and also acute or chronic inflammatory diseases,and more generally, diseases linked to the overproduction of ILβ or TNFαcytokines.

2. Description of the Related Art

Active anti-cytokine immunization is an active immunotherapy strategydeveloped since 1990 by Zagury et al. which is based in particular onPatent Application WO 92/22577. This idea was taken up by severalscientific teams which have published in international scientificjournals, active immunizations against the entire IFNα proteinmultimerized by treatment with glutaraldehyde (Gringeri et al., JAIDS1999; 20:358-70), a chimeric TNFα protein consisting of coupling thenative TNFα protein with a T epitope of ovalbumin (Dalum at al., NatureBiotechnology, 1999; 17:666-69), against entire IL9 coupled with KLH(Richard et al., PNAS, 2000; 97:767-72) or also chimeric entire IL5 witha T epitope of tetanus toxin (Hertz et al., J. Immunol, 2001;167:3792-99).

These approaches have confirmed the feasibility of autologousanti-cytokine immunizations, but these few successes obscure theunsuccessful tests described by certain authors: certain cytokines donot allow sufficiently protective and clinically effective antibodies tobe obtained, and the same cytokine prepared in a form which is effectivein one manner, will not be effective in another (Richard et al., PNAS,2000; 97:767-72).

In trying to explain this phenomenon, the Applicant has observed that todate all the authors have used entire cytokines (optionally slightlymodified), which leads to difficulties in particular at the followinglevels:

-   -   dilution of the immunogenic power of the antigenic determinants        of interest    -   possible genesis of facilitating antibodies in vivo (E        response).    -   possible genesis of autoimmune reaction to the potential T        epitopes present in the entire cytokine (autoimmune T reaction).

This is why the Applicant has previously claimed families of peptides oflimited size between 5 and 40 amino acids originating from cytokines andwhich have an antigenic power making it possible to generate antibodiesagainst the native cytokine (Patent Application PCT/FR03/01120).EP-A-0218531 also described IL1 peptides used for the preparation ofantibodies.

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

SUMMARY OF THE INVENTION

The Applicant has evaluated certain peptides of the IL1β, and TNFcytokines with a size comprised between 10 and 30 residues and hasdemonstrated that these peptides were not only antigenic, but that theywere also effective as immunogens for protecting in vivo againstdiseases linked to the overproduction of these cytokines. The presentApplication therefore claims peptides of a size comprised between 5 and30 amino acids, originating from murine or human IL1β and TNFα cytokinesor those of any other species of mammal, and their use in humans oranimals (in this case veterinary use) for preventing or treatingdiseases linked to the overproduction of these cytokines. The presentApplication also claims the production of monoclonal or oligoclonalantibodies from these peptides and the use of these antibodies fortherapeutic or preventive administration to humans or animals(veterinary application).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing survival of mice immunized against peptidesand subjected to septic shock.

FIG. 2 is a graph showing average clinical scores of groups of miceimmunized against peptides in a model of collagen arthritis.

DETAILED DESCRIPTION OF THE INVENTION

The cytokine peptides according to the invention originate from or arederived from the IL1β and TNFα cytokines. By “originate” is meant thattheir amino acid sequence is identical to that of the cytokine. By “arederived” is meant that their amino acid sequence is mostly identical tothat of the cytokine but can comprise a few differences as will be seenhereafter.

The above cytokine peptides advantageously comprise more than 5, inparticular more than 7, particularly more than 9 and quite particularlymore than 11 amino acids.

Under other preferential conditions for implementing the invention, theabove cytokine peptides comprise less than 30, advantageously less than25, in particular less than 20, more particularly less than 18 aminoacids.

The Applicant has demonstrated that the peptide area:

80 VSRFAISYQEKVNLLS 95 (SEQ ID NO:1) of the murine TNFα cytokine makesit possible to engender antibodies by active immunization, capable ofblocking TNFα-dependent septic shock in mice (Experiment 1). Thecorresponding sequence of the human cytokine is 75% homologous:

80 ISRIAVSYQTKVNLLS 95 (SEQ ID NO: 2)

The Applicant has also demonstrated that the peptide area:

121 YISTSQAEHKPVFLG 135 (SEQ ID NO:3) of the murine IL1β cytokine makesit possible to engender antibodies by active immunization, capable ofblocking collagen arthritis induced in mice (Experiment 2). Thecorresponding sequence in humans is 80% homologous:

121 YISTSQAENMPVFLG 135 (SEQ ID NO: 4)

Loetasoher et al. (J Biol Chem, 1993, 268:26350-357) have shown by sitespecific mutagenesis experiments that the 143-147 region of TNFα couldbe important in the interaction with the p55 receptor of TNFα, and thepresent invention also claims the immunization against B epitopes ofthis region by means of peptides having a significant homology, be theyxenogenic or mutant in relation to the human or animal cytokinesequence. The sequence corresponding to this area in humans is:

140 DYLDFAESGQVY 150 (SEQ ID NO: 5)

and in mice:

140 KYLDFAESGQVY 150 (SEQ ID NO: 6)

Evans et al. Biol Chem, 1995, 270:11477-83) have also explained by sitespecific mutagenesis experiments that other regions of IL1β could beimportant in the interaction with the receptor, and the presentinvention also claims immunization against B epitopes of these regionsby means of peptides having a significant homology, be they xenogenic ormutants in relation to the targeted human or animal cytokine sequence.The sequences corresponding to this area in humans are:

  3 VKSLNCTLRDSQQKSL 18 (SEQ ID NO: 7)  45 SFVQGEESNDKIP 57(SEQ ID NO: 8)  89 NYPKKKMEKRFVFNKIEI 106 (SEQ ID NO: 9)143 ITDFTMQFVSS 153 (SEQ ID NO: 10)

and in mice:

  3 IRQLHYRLRDEQQKSL 18 (SEQ ID NO: 11)  45 SFVQGEPSNDKIP 57(SEQ ID NO: 12)  89 QYPKKKMEKRFVFNKIEV 106 (SEQ ID NO: 13)142 IIDFTMESVSS 152 (SEQ ID NO: 14)

It has been possible to create autologous antibodies against the latterpeptides of mice by active immunization.

Experiments 1 and 2 hereafter, carried out by the Applicant, demonstratethe therapeutic ability in vivo of the anti-peptide active immunizationapproach with peptides of a size less than 30 residues. The Applicantclaims these peptides and their derivatives for also carrying out activeimmunizations allowing the generation of antibodies targeting theseepitopes of the TNFα and IL1b cytokines in order to block theinteraction of the cytokine with its receptor.

As is known by a person skilled in the art of immunology, modificationsof the natural peptide chains are possible without however modifying thenature of the immunological properties of the immunogenic peptides.Derivatives of cytokine peptides can therefore also be mentioned, whichare highly homologous to these natural sequences, for example havingmore than 50% homology, in particular more than 70% homology, andpreferably more than 80% homology or even more than 90% homology withthe corresponding native peptide whilst retaining the immunologicalproperties of this epitopic site of the native peptide. In particularhomologous sequences of cytokines of other mammals can be used in orderto obtain a xenogenic and not only an autologous protective reaction inan animal or human.

The cytokine peptide derivatives can contain modified residues, oncondition that the modifications do not appreciably reduce theimmunogenicity, either by adding chemical radicals (methyl, acetyl etc.)or by stereochemical modification (use of D series amino acids). Thecytokine peptide derivatives should, like the cytokine peptides induceantibodies interacting with cytokine.

The cytokine peptide derivatives according to the invention can compriseone or more modifications in the amino acids of which they areconstituted, such as deletions, substitutions, additions, orfunctionalizations (such as acylation) of one or more amino acids, tothe extent that these modifications remain within the frameworkspecified above (immunological characters). For example, in general thereplacement of a leucine residue by an isoleucine residue does notmodify such properties; the modifications should generally concern lessthan 40% of the amino acids, in particular less than 30%, preferablyless than 20% and quite particularly less then 10% of the amino acids ofthe natural peptide. It is important that the antibodies induced by themodified peptides are active vis-à-vis native cytokine.

These modifications are within the scope of a person skilled in the art,who can verify the incidence of the modifications by simple tests. Theimmunogenicity of such modified derivatives can be evaluated by ELISAafter immunization of mice, the antigen tested by ELISA being the entirecytokine or the immunizing cytokine peptide, or by cytokine-receptorbond blocking tests. The possible modifications preferably affect lessthan 8 amino acids, advantageously less than 6 amino acids, inparticular less than 4 amino acids, and particularly 3 amino acids orless, such as 2 or 1 single amino acid.

A subject of the invention is also a compound characterized in that itcontains at least one abovementioned cytokine peptide or cytokinepeptide derivative. Such a compound can comprise identicalpeptide/derivative repetitions, or different peptide/derivativecombinations, either in linear form or in the form of a candelabrastructure or couplings mixed with carrier proteins. Such a compound canalso be presented in cyclized form. Thus cytokine peptides or cytokinepeptide derivatives according to the invention can for example beinserted into longer sequences of amino acids providing in particular abetter conformation or combined with exogenous T epitopes (whether forprotein or DNA immunizations).

They can advantageously be associated in a covalent manner with carrierproteins such as for example KLH.

The cytokine peptides according to the invention, due to their limitedsize, correspond in general to a small number of epitopes of thecytokine. When they are in particular inserted, combined or associated,the above compounds do not comprise other epitopes of said cytokine.

These cytokine peptides or cytokine derivatives can be included in anyprotein sequence which does not comprise homology with the other naturalcytokine epitopes. For example, a cysteine can be added to the ends inorder to confer a cyclic structure on the peptide. Another example is apeptide surrounded by sequences of T epitopes of the tetanus toxin. Yetanother example can comprise a peptide corresponding to the sequence ofthe receptor binding site but where certain amino acids are replaced bytheir D series isomers in order to avoid their agonist effect. In fact,it can be optionally advantageous to use peptide derivatives which haveno agonist activity on the receptor so that the immunogen does notinterfere with the immune response.

In order to increase the immune response, these cytokine peptides orcytokine derivatives can be coupled to carrier proteins. The couplingmethods and the carrier protein considered can be different according tothe target peptide: they can for example be Keyhole Limpet Hemocyanin(KLH) protein and Tetanus Toxoid (TT) conjugated to the peptides bychemical methods well known to a person skilled in the art such as thoseof carbodiimide, glutaraldehyde or bis-diazotized benzidine coupling.The realization of these couplings can be facilitated by the addition orincorporation of amino acids into the sequence, such as for examplelysines, histidines, tyrosines or cysteines. Such peptide compoundscoupled to an exogenous T epitope (originating from plasmodiumfalciparum, KLH, etc.) whether chemically or genetically also comewithin the scope of the invention.

Network couplings of candelabra type or to molecules such as transferrinor ferritin can also be implemented in order to effectively stimulatethe immune response.

The peptides according to the invention can in particular be produced bychemical synthesis or genetic engineering or any other suitable method.The synthesis of cyclic peptides, grafting, as needed, one or more aminoacids at the end of the chain as cysteines in order to create adisulphide bridge makes it possible to recover part of the secondarystructure that these peptide fragments possess in the three-dimensionalstructure of the protein.

The peptides according to the invention possess very usefulpharmacological properties. In particular they possess remarkableanti-cytokine properties. These properties are illustrated hereafter inthe experimental part. They justify the use of the peptides describedabove as a medicament.

This is why a subject of the invention is also medicaments characterizedin that they are constituted by peptides or derivatives of the IL1β orTNFα cytokines or compounds as defined above, i.e. cytokine peptides orcytokine derivatives or immunogenic compounds as defined above for theiruse in a method of therapeutic treatment of the human or animal body, aswell as the use of such a cytokine peptide or cytokine derivative orimmunogenic compound for the preparation of a curative or preventivemedicament intended for the treatment or prevention of diseases linkedto an excess or to the presence of cytokines.

The medicaments according to the present invention are used for examplein both the curative and preventive treatment of diseases linked tocytokine deregulation, whether rheumatoid polyarthritis, septic shock orany other disease where the blocking of IL1β or TNFα is curative. Theseare only a few examples, and a subject of the invention is also anytreatment of the human or animal body based on active immunization (DNAor peptide) involving the peptide sequences mentioned above to theexclusion of other epitopes of the cytokines. These sequences can bemodified as indicated in the present description, and the immunizationsby DNA are carried out by simple translation from the genetic code.

The humoral immunity response can be evaluated by ELISA tests.

The immunogenic active ingredients according to the invention can beused as follows:

A cytokine peptide or cytokine derivative or immunogenic compoundaccording to the present invention, is administered to a patient or toan animal, for example by sub-cutaneous or intramuscular route, in asufficient quantity to be effective at a therapeutic level, to a subjectneeding such treatment. The dose administered can for example range from1 to 1000 μg, in particular 10 to 500 μg, by sub-cutaneous route, once amonth for three months, then periodically as a function of the inducedserum antibodies count, for example every 2-6 months. In the samepreparation two immunogenic molecules of the two cytokines can beadministered if a still stronger blocking effect is to be obtained.

A subject of the invention is also the pharmaceutical compositions inparticular the vaccines which contain at least one abovementionedcytokine peptide or cytokine derivative or immunogenic compound, asactive ingredient.

As medicaments, a cytokine peptide or cytokine derivative or immunogeniccompound of the invention can be incorporated into pharmaceuticalcompositions intended for any standard route in use in the field ofvaccines, in particular by sub-cutaneous route, by intramuscular route,by intravenous route or by oral route. The administration can take placein a single dose or repeated once or more after a certain period oftime.

This is why a subject of the present Application is also a curative orpreventive pharmaceutical composition, characterized in that itcomprises as active ingredient, one or more cytokine peptides orcytokine derivatives or immunogenic compounds, as defined above.

The immunogenic agent can be conditioned alone or mixed with anexcipient or mixture of pharmaceutically acceptable excipients as anadjuvant. A subject of the present Application is more particularly avaccine containing as immunogen, an abovementioned cytokine peptide orcytokine derivative or immunogenic compound.

A subject of the present invention is also a process for preparing acomposition described above, characterized in that, according to methodsknown per se, the active ingredient or ingredients are mixed withacceptable, in particular pharmaceutically acceptable excipients.

The administration to a patient of a cytokine peptide or cytokinederivative or immunogenic compound according to the inventioncorresponds to an active immunotherapy.

It can also be useful to carry out passive immunotherapy, i.e. toprovide a patient or a sick animal directly with the antibodies whichthey need. For this purpose, the peptides, derivatives and compoundsdefined previously can be used in order to produce monoclonal antibodiesaccording to the usual techniques, human, murine or humanized, forexample by conversion of B lymphocytes from a subject immunized by theEpstein-Barr virus or by the screening of antibody libraries. Theseantibodies by targeting the epitopes of the above peptides block theinteraction of the cytokine with its receptor and thus make it possibleto reduce the pathogenic effect of the cytokine in the disease.Oligoclonal antibodies can also be prepared by active immunization inanimals such as horses for example, purified, and administeredtherapeutically to humans or animals.

The vaccine preparations can be packaged for the intra-nasal route inthe form of gel with carbopol as excipient, nasal drops or spray and forthe oral route in the form of gastroresistant capsules, sugar-coatedtablets or gastroresistant granules.

In the case of DNA vaccine administered by systemic or mucosal route,the galenic presentation of the plasmid can be a suspension in aphysiological liquid such as physiological PBS (phosphate bufferedsaline=PBS). The plasmids can be enclosed in biodegradable polymer (PLG,PLA, PCL) microspheres and administered in gastroresistant capsules foringestion (oral route). The DNA can also be expressed in a bacterial,salmonella-type or viral-type, adenovirus or poxvirus living vector.

Finally, a subject of the present Application is a process for activeimmunization of patients characterized in that as immunogen, a cytokinepeptide or cytokine derivative or immunogenic compound is used, asdefined above, advantageously associated with a mineral, oily orsynthetic immunity adjuvant.

The immunizations can be done in a standard fashion in particular bypeptides or immunogenic compounds as conjugates preferably in thepresence of an adjuvant, for example ISA 51 or Alum. The immunizationscan be DNA-based (sequences homologous to the binding sites combinedwith exogenous T epitopes) using naked DNA or an expression vectorcontaining an adapted promoter such as for example pCR3.1. The DNAsadministered can be protected from the nucleases by the use ofappropriate radicals (CpG etc.). In particular an initial DNAimmunization can be followed by standard boosters using the peptidecompounds.

The methods of treatment of the human or animal body described in thispatent can include a cytokine peptide or cytokine derivative orimmunogenic compound as defined above, and can include the monoclonal oroligoclonal antibodies as defined above.

The preferential conditions for using the peptides described above alsoapply to the other subjects of the invention referred to above.

FIG. 1 and FIG. 2 show the results of protection in vivo of theimmunizations described in Experiments 1 and 2 for a TNFα peptide inTNFα-dependent septic shock and an IL1β peptide in collagen arthritis.

The experiments which follow illustrate the present invention.

Experiment 1

4 murine TNFα peptides coupled to the KLH carrier protein, including thecyclized peptide CVSRFAISYQEKVNLLSC (SEQ ID NO:15) called TNFα-6, weretested in a model of endotoxinic shock. For this purpose, Balb/c micewere preimmunized against these peptides on days D0, D0, D16, and D40.On day D50, the mice were subjected to shock, i.e. they were injectedwith LPS with Galactosamine. A control was carried out on mice immunizedagainst KLH alone.

It is noted that all the mice died after two days, except for the groupimmunized against the TNFα-6 peptide where the mice were protected. Theprotection conferred by the immunization was very significant (p=0.008).

Experiment 2

3 murine IL1β peptides coupled to the KLH protein, including thecyclized IL1-6 peptide of sequence YCYISTSQAEHKPVFLGC (SEQ ID NO:16),were tested in a model of collagen arthritis. For this purpose, DBA1mice were preimmunized against these peptides on days D0, D20, D40, DG0.On day D80 the mice were immunized against collagen in Freund'sadjuvant, and similarly on day D95. The development of arthritis wasmonitored between day D100 and day D160: twice weekly, the mice wereexamined and a score was attributed to them as a function of the stateof inflammation of their joints (0=no inflammation, 1=slightinflammation, 2=average inflammation, 3=strong inflammation).

It is noted that the control mice immunized by KLH alone exhibit stronginflammation (curve with squares) whereas the group of mice immunizedagainst the IL1β-6 peptides (curve with diamonds) exhibit a strongprotection level with respect to the control (p=0.0003, ANOVA test).

Experiment 3

6 groups of 4 mice were immunized against 20 ug of the murine IL1βpeptides IRQLHYRLRDEQQKSL (group 1) (SEQ ID NO:11), SFVQGEPSNDKIP (group2) (SEQ ID NO:12), QYPKKKMEKRFVFNKIEV (group 3) (SEQ ID NO:13),IIDFTMESVSS (group 4) (SEQ ID NO:14), and 20 ug of the murine TNFαpeptide KYLDFAESGQVY (group 5) (SEQ ID NO:6) all coupled to KLH. Group 6corresponds to mice immunized by KLH alone. A booster was administeredon days D20 and D40. On day D50 the mice were sacrificed and theantibodies directed against the native mTNFα cytokine and the nativemIL1β cytokine in the serums were measured by an ELISA test.

The averages obtained for each group are indicated in the tablehereafter.

TABLE 1 Anti-TNFα response Anti-IL1β response Average Average Group 10.13 1.5 Group 2 0.15 1.9 Group 3 0.18 1.6 Group 4 0.12 0.8 Group 5 1.30.31 Group 6 0.10 0.15

It is therefore clear that these peptides are well capable of inducingantibodies recognizing the native cytokine. These antibodies are veryspecific since they recognize only the cytokine the sequence of whichwas used for the immunization.

What is claimed is:
 1. An isolated immunogenic compound consisting of(a) a the peptide of less than 20 residues in length that comprises anamino acid sequence with more than 90% sequence identity to SEQ ID NO:7,coupled to (b) a heterologous carrier protein that increases theimmunogenicity of the peptide in (a).
 2. The immunogenic compound ofclaim 1, wherein the peptide is the human IL1β cytokine peptideconsisting of the sequence of SEQ ID NO:7.
 3. The immunogenic compoundof claim 1, wherein the peptide of (a) is coupled to the heterologouscarrier protein of (b) by chemical conjugation.
 4. The immunogeniccompound of claim 1, wherein the heterologous carrier protein is KeyholeLimpet Hemocyanin.
 5. The immunogenic compound of claim 1, wherein theheterologous carrier protein is Tetanus Toxoid.
 6. The immunogeniccompound of claim 1, wherein one or more amino acids of said peptide aremodified by cyclization, stereochemical modification orfunctionalization of one or more amino acids of said peptide.
 7. Theimmunogenic compound of claim 6, wherein the functionalization isacylation.
 8. An immunizing composition, comprising the immunogeniccompound of claim 1 and an adjuvant.
 9. An immunogenic compoundconsisting of (a) a peptide of less than 30 residues in length thatcomprises an amino acid sequence with more than 90% sequence identity tothe amino acid sequence of SEQ ID NO:8, coupled to (b) a heterologouscarrier protein that increases the immunogenicity of the peptide in (a).10. The immunogenic compound of claim 9, wherein the peptide is thehuman IL1β cytokine peptide consisting of the sequence of SEQ ID NO:8.11. The immunogenic compound of claim 9, wherein the peptide is lessthan 25 residues in length.
 12. The immunogenic compound of claim 9,wherein the peptide is less than 20 residues in length.
 13. Theimmunogenic compound of claim 9, wherein the peptide of (a) is coupledto the heterologous carrier protein of (b) by chemical conjugation. 14.The immunogenic compound of claim 9, wherein the heterologous carrierprotein is Keyhole Limpet Hemocyanin.
 15. The immunogenic compound ofclaim 9, wherein the heterologous carrier protein is Tetanus Toxoid. 16.The immunogenic compound of claim 9, wherein one or more amino acids ofsaid peptide are modified by cyclization, stereochemical modification orfunctionalization of one or more amino acids of said peptide.
 17. Theimmunogenic compound of claim 16, wherein the functionalization isacylation.
 18. An immunizing composition, comprising the immunogeniccompound of claim 9 and an adjuvant.